A staphylococcal radioimmunoassay for detection of antibodies toMycoplasma pneumoniae

A radioimmunoassay (RIA) which depends on the property of protein A ofStaphylococcus aureus to combine with the Fc-fragment of immunoglobulins was developed.This technique was employed to measure antibodies in human and various animal sera. It could be demonstrated that the staphylococcal RIA was at least as sensitive as the previously described radioimmunoprecipitation technique in detecting antibodies toM.pneumoniae in human sera. In addition, antibodies toM.pneumoniae could be demonstrated in sera of hamsters intranasally inoculated with the organisms. Antibodies could also be demonstrated in rabbit sera after immunization withM.pneumoniae. The test proved to be considerably more sensitive than conventional tests for detection of antibodies to the organisms. The test requires only small amounts of reagents and is relatively inexpensive.The results were presented in a preliminary form at the annual meeting of the Local Branch of the American Society for Microbiology, Frankfurt, March 1976 and the 77th ordinary meeting of the Society for General Microbiology, Glasgow, September 1976     

Methylenetetrahydrofolate reductase and thymidylate synthase genotypes and risk of acute graft-versus-host disease following hematopoietic cell transplantation for chronic myelogenous leukemia.

Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) play key roles in intracellular folate metabolism. Polymorphisms in these enzymes have been shown to modify toxicity of methotrexate (MTX) after hematopoietic cell transplantation. In this study, we evaluated the risk of acute graft-versus-host disease (GVHD) associated with genetic variation in recipient and donor MTHFR and TS genotypes to assess whether genotype alters the efficacy of MTX in acute GVHD prophylaxis. Data on the transplantation course were abstracted from medical records for 304 adults who received allogeneic hematopoietic cell transplants. MTHFR (C677T and A1298C) and TS (enhancer-region 28-base pair repeat, TSER, and 1494del6) genotypes were determined using polymerase chain reaction/restriction fragment length polymorphism and TaqMan assays. Multivariable logistic regression was used to assess the associations between genotypes and risk of acute GVHD. Compared with recipients with the wild-type MTHFR 677CC genotype, those with the variant 677T allele showed a decreased risk of detectable acute GVHD (677CT: odds ratio, 0.8; 95% confidence interval, 0.4-1.6; 677TT: odds ratio, 0.4; 95% confidence interval, 0.2-0.8; P for trend = .01). The variant MTHFR 1298C allele in recipients was associated with an increased risk of acute GVHD compared with the wild-type MTHFR 1298AA genotype (1298AC: odds ratio, 2.0; 95% confidence interval, 1.1-3.9; 1298CC: odds ratio, 3.6; 95% confidence interval, 1.0-12.7; P for trend < .01). No association with risk of acute GVHD was observed for donor MTHFR genotypes or for recipient or donor TS genotypes, with the exception of an increase in acute GVHD among recipients whose donors had the TSER 3R/2R genotype (odds ratio, 3.0; 95% confidence interval, 1.3-7.2). These findings indicate that host, but not donor, MTHFR genotypes modify the risk of acute GVHD in recipients receiving MTX, in a manner consistent with our previously reported associations between MTHFR genotypes and MTX toxicity. A direct trade-off between drug toxicity and drug efficacy may play a role. Alternatively, the systemic folate environment, regulated by host tissues, might influence donor T-cell growth and activity.     

Proton magnetic resonance spectroscopy in ecstasy (MDMA) users

The popular recreational drug 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has well-recognized neurotoxic effects upon central serotonergic systems in animal studies. In humans, the use of MDMA has been linked to cognitive problems, particularly to deficits in long-term memory and learning. Recent studies with proton magnetic resonance spectroscopy (1H MRS) have reported relatively low levels of the neuronal marker N-acetylaspartate (NAA) in MDMA users, however, these results have been ambiguous. Moreover, the only available 1H MRS study of the hippocampus reported normal findings in a small sample of five MDMA users. In the present study, we compared 13 polyvalent ecstasy users with 13 matched controls. We found no differences between the NAA/creatine/phosphocreatine (Cr) ratios of users and controls in neocortical regions, and only a tendency towards lower NAA/Cr ratios in the left hippocampus of MDMA users. Thus, compared with cognitive deficits, 1H MRS appears to be a less sensitive marker of potential neurotoxic damage in ecstasy users.     

Escherichia coli Nissle 1917 distinctively modulates T-cell cycling and expansion via toll-like receptor 2 signaling

Although the probiotic Escherichia coli strain Nissle 1917 has been proven to be efficacious for the treatment of inflammatory bowel diseases, the underlying mechanisms of action still remain elusive. The aim of the present study was to analyze the effects of E. coli Nissle 1917 on cell cycling and apoptosis of peripheral blood and lamina propria T cells (PBT and LPT, respectively). Anti-CD3-stimulated PBT and LPT were treated with E. coli Nissle 1917-conditioned medium (E. coli Nissle 1917-CM) or heat-inactivated E. coli Nissle 1917. Cyclin B1, DNA content, and caspase 3 expression were measured by flow cytometry to assess cell cycle kinetics and apoptosis. Protein levels of several cell cycle and apoptosis modulators were determined by immunoblotting, and cytokine profiles were determined by cytometric bead array. E. coli Nissle 1917-CM inhibits cell cycling and expansion of peripheral blood but not mucosal T cells. Bacterial lipoproteins mimicked the effect of E. coli Nissle 1917-CM; in contrast, heat-inactivated E. coli Nissle 1917, lipopolysaccharide, or CpG DNA did not alter PBT cell cycling. E. coli Nissle 1917-CM decreased cyclin D2, B1, and retinoblastoma protein expression, contributing to the reduction of T-cell proliferation. E. coli Nissle 1917 significantly inhibited the expression of interleukin-2 (IL-2), tumor necrosis factor alpha, and gamma interferon but increased IL-10 production in PBT. Using Toll-like receptor 2 (TLR-2) knockout mice, we further demonstrate that the inhibition of PBT proliferation by E. coli Nissle 1917-CM is TLR-2 dependent. The differential reaction of circulating and tissue-bound T cells towards E. coli Nissle 1917 may explain the beneficial effect of E. coli Nissle 1917 in intestinal inflammation. E. coli Nissle 1917 may downregulate the expansion of newly recruited T cells into the mucosa and limit intestinal inflammation, while already activated tissue-bound T cells may eliminate deleterious antigens in order to maintain immunological homeostasis.     

Histopathological studies on the local reactions induced by complete Freund's adjuvant (CFA), bacterial lipopolysaccharide (LPS), and synthetic lipopeptide (P3C) conjugates

The inflammatory reactions following subcutaneous application of adjuvants revealed characteristic pathological patterns. The injection of complete Freund's adjuvant (CFA) resulted in the formation of large lipid deposits encircled by an inflammatory reaction and concentrically arranged collagen bundles. Bacterial lipopolysaccharide (LPS) caused granulomatous aggregations of mononuclear cells with thrombotic vessel occlusions. Inoculation of the lipopeptide adjuvants induced accumulation of mononuclear cells with only minimal fibrotic changes which were resolved after day 28. Lipopeptide conjugates based on the head group tripalmitoyl-S-glyceryl-cysteinyl-serin (P3CS) can thus be used as effective immunogens and adjuvants without long-term tissue damage.     

Sodium gradient-energized concentrative transport of adenosine in renal brush border vesicles

The uptake of adenosine in brush border vesicles of the proximal tubule of the rat kidney has been studied with a filtration technique. The initial rate of uptake was almost 6 times greater in the presence of NaCl than in the presence of KCl. The stimulatory effect of Na⁺ was strictly dependent on a gradient of Na⁺ (out>in). The time course of uptake showed an overshoot with a maximum at 20 s with a gradient of NaCl, but not with KCl. Inosine and 5-AMP were produced from adenosine within the vesicles. In the presence of an inhibitor or adenosine deaminase adenosine was not significantly metabolized during the first 20 s of uptake. Thus, kinetic parameters of transport could be studied in the absence of interferences with metabolism. AK _m of 1.1 M and aV _max of 232 pmol · min^–1 · mg protein^–1 were calculated for the Na⁺ gradient-dependent transport. The dependency on a Na⁺ gradient, the capacity for uphill transport and the high affinity for adenosine situate this transport system apart from the mechanisms of transport of nucleosides described so far. It may be relevant in regard to the role of adenosine in the regulation of glomerular filtration.Abbreviations used EHNA erythro-9-(2-hydroxy-3-nonyl)adenine - FCCP carbonylcyanide p-trifluoromethoxy-phenylhydrazone - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Tris tris (hydroxymethyl)-aminomethane     

Acquisition of vimentin in astrocytes cultured from postnatal rat brain

Summary Vimentin and glial fibrillary acidic protein (GFAP) represent the principal constituents of intermediate filaments found in astrocytes. In contrast to vimentin—GFAP transition which occurs during glial developmentin situ, vimentin coexists with GFAP in cortical astrocytes allowed to differentiate in culture. To examine whether culture conditions or proliferative activity of the cells is responsible for the expression of vimentin, we generated cultures of GFAP-positive, vimentin-negative astrocytes isolated from 26-day postnatal rat brain cortices. Isolated astrocytes are characterized by a very thin rim of perinuclear cytoplasm and by numerous processes. Antiserum to GFAP labelled major processes and cell somata of some astrocytes, especially those with relatively short and large processes. Within 3 days in culture, all astrocytes accumulated GFAP in hypertrophic cell bodies and many began to express vimentin. Vimentin appeared primarily close to nuclei, and filaments of vimentin extended into proximal segments of the cell processes. In some astrocytes, however, vimentin was always absent. Combined double immunolabelling and histoautoradiography experiments demonstrated that the acquisition of vimentin was independent of the ability of astrocytes to incorporate tritiated thymidine. The results indicate that astrocytes isolated from 26-day postnatal rat brain are heterogeneous with respect to their ability to express vimentin and that vimentin synthesis is not correlated with the growth state of the cells as had been previously suspected.     

Somatoform pain disorder in the general population

BACKGROUND: Chronic pain disorder is assumed to represent a frequent and disabling condition. However, data on the prevalence of somatoform pain symptoms and somatoform pain disorder in the community are limited to date. METHODS: German versions of the Composite International Diagnostic Interview were administered to a representative national sample of 4,075 people. Somatoform pain disorder was diagnosed by standardized diagnostic algorithm based on the DSM-III-R criteria (absence of adequate physical findings). One subgroup was identified as also meeting the DSM-IV criterion B for 'significant distress or psychosocial impairment due to the somatoform pain'. RESULTS: A lifetime prevalence rate of somatoform pain disorder according to DSM-III-R of 33.7% and a 6-month rate of 17.3% was found. When applying the DSM-IV B criterion, the prevalence rate dropped to 12.3 and 5.4%, respectively. In both groups more than 95% of the probands had contacted their doctor because of the pain. In 25% of the probands the pain was positively assigned to psychological factors. A female:male ratio of 2:1 was found. CONCLUSIONS: Somatoform pain disorder (DSM-III-R) is a frequent condition. However, only about one third of these subjects is severely distressed or impaired by the pain. A clear operationalized concept of the DSM-IV criterion C 'psychological factors are judged to have an important role in the onset, severity, exacerbation or maintenance of the pain' should be provided in the further development of the diagnosis 'pain disorder' in order to make this diagnosis suitable for general population surveys.     

A modified Sindbis vector for prolonged gene expression in neurons

Sindbis viruses have been widely used in neurobiology to express a variety of genes in cultured neurons, in cultured slices, and in vivo. They provide fast onset and high levels of expression of foreign genes, but the expression is limited to a short time window due to a shut-off of host protein synthesis. We have used a mutation in an essential gene (nsP2) of the life cycle of Sindbis, which allows the functional analysis of changes in protein expression for >/=6 days after infection. This Sindbis mutant (nsP2) was used to express enhanced green fluorescent protein (EGFP) in hippocampal neurons in culture and in vivo without any sign of toxicity, based on two-photon imaging and electrophysiology. In addition, the EGFP mutant virus can be injected in vivo to visualize spines and other details of neuronal structure. The Sindbis mutant described here provides an improved tool in neurobiology with reduced cytotoxicity and a prolonged time window of expression for novel applications in imaging and behavior. In addition, the use of this vector for the functional expression of mammalian voltage-gated ion channels in organotypic slices is demonstrated.     

Endotoxins prevent murine IgE production,T(H)2 immune responses,and development of airway eosinophilia but not airway hyperreactivity

BACKGROUND: Contact with immunomodulatory factors, such as LPS, in early infancy is associated with decreased allergen sensitization. OBJECTIVE: We sought to study the effects of systemic or airway exposure with LPS on the development of allergen sensitization, eosinophilic airway inflammation, and increased in vivo airway reactivity (AR) in a mouse model. METHODS: BALB/c mice were systemically sensitized with ovalbumin (OVA) plus adjuvant on days 1 and 14 and challenged through the airways with allergen on days 34 to 36. We performed measurement of OVA-specific IgE serum levels, in vitro T(H)2 cytokine production, differential cell counts in bronchoalveolar lavage fluids, and assessment of in vivo AR to inhaled methacholine by means of barometric whole-body plethysmography. RESULTS: Systemic LPS administration before OVA sensitization reduced OVA-specific IgE serum levels (426 +/- 76 vs 880 +/- 104 U/mL, P <.01), T(H)2 cytokine production by splenic mononuclear cells (IL-4: 0.08 +/- 0.01 vs 0.17 +/- 0.01 ng/mL; IL-5: 1.98 +/- 0.52 vs 4.11 +/- 0.54 ng/mL; P <.01), and extent of airway eosinophilia (total cell counts: 93 vs 376 x 10(3)/mL; eosinophils: 23% vs 51%; P <.01) compared with that in OVA-sensitized mice. Local LPS administration to sensitized mice before airway allergen challenges particularly induced IFN-gamma production by peribronchial lymph node cells in vitro (1718 +/- 315 vs 483 +/- 103 ng/mL, P <.01) associated with reduced airway eosinophilia compared with that seen in OVA-sensitized mice. Development of increased AR was not affected by systemic or local LPS exposure. Inhibitory effects of LPS on allergen sensitization and eosinophilic airway inflammation were inhibited by administration of anti-IL-12 antibodies before LPS exposure. CONCLUSION: These data indicate that local and systemic application of LPS modulates systemic and local T(H)1/T(H)2 immune responses in a distinct but similarly IL-12-dependent mode.